The present invention is concerned with a method for generating, in a non specific manner, multiple copies of RNA from a pool of mRNA""s. Such a method is of particular importance in techniques for screening the differences in expression in given cell types or in cells under specific conditions.
In cells of higher organisms only some 15% of the genes present (each cell contains about 100,000 genes) is expressed. Gene expression varies between different cell types and between different stages of development of a given cell and is crucial to all biological processes, such as aging, cell differentiation, and infectious or other disease states. Thus the identification of genes that are differentially expressed in cells under different conditions is of prime interest in cellular biology.
To be able to analyze the mRNA content derived from only a few cells a method is needed to amplify the mRNA present in the cell(s) under investigation. Much effort has already been put in methods to examine the mRNA population of a cell. This has lead to the development of techniques to label nucleic acid material starting from the mRNA population of a cell aimed at the identification of genes that are differentially expressed in cells under various conditions. One method for screening differences in gene expression is a method known as Differential Display (Liang and Pardee, Science, Vol 257, 967-971, 1992; U.S. Pat. No. 5,262,311 which issued on Nov. 16, 1993). With the method of Liang and Pardee mRNA is first transcribed into cDNA and amplified using the Polymerase Chain Reaction (PCR). A set of oligonucleotide primers is used, the first of which is anchored to the polyadenylated tail of a subset of mRNA""s, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer. The method is used with different pairs of alterable sequences aiming at the amplification of as many mRNA""s as possible from the cells under investigation. The PCR products are labeled using tracer amounts of labeled (radioactive) nucleotides.
An improvement on the Differential Display method of Liang and Pardee was disclosed in U.S. Pat. No. 5,589,726. The method described in U.S. Pat. No. 5,589,726 differs from the method of Liang and Pardee in that it uses longer primers (22-30 nucleotides compared to the 9-14 base primers originally described by Liang and Pardee).
Another alleged improvement over the Differential Display technique as originally disclosed by Liang and Pardee is disclosed in WO 97/37045. In this application a method is disclosed that, again, is based on PCR: This method uses an oligo-dT primer rather than an anchored primer. Thus, after the reverse transcription step only one cDNA population covering all possible mRNA sequences is created. The cDNA thus obtained is titrated into the PCR process by running several IPCR reactions at decreasing concentrations of cDNA. This serves to calibrate the method and to protect it against false negatives. The PCR reaction may be performed with anchored primers again.
Yet another method for xe2x80x9cexpression profilingxe2x80x9d of mRNA""s is disclosed in U.S. Pat. No. 5,514,545. With this method mRNA in a single cell can be characterized by microinjecting into a cell a first amplification oligonucleotide comprising oligo-dT and the sequence of a bacteriophage promoter such as T7, T3 or SP6, reverse transcriptase and nucleotides to synthesize a first strand of cDNA from the mRNA in the cell. From the first strand of cDNA double stranded cDNA is synthesized. Since this double stranded cDNA includes a functional promoter aRNA (anti-sense RNA) can now be synthesized therefrom using an RNA polymerase. The aRNA is now reamplified using random hexanucleotide primers with a reverse transcriptase to form first strand cDNA.
With all the above techniques cDNA is made starting with a primer using the mRNA as a template. However, the enzyme that is used for this reaction (reverse transcriptase) is hampered in the cDNA synthesis by structures in the mRNA. As a result the prior art methods are selective for mRNA""s with little or no structure. This effect is further enhanced if the synthesized cDNA is amplified further, for instance by PCR. Due to the aforementioned it is common practice to use a large sample amount in these type of expression profiling analysis. Thus this technical threshold does not allow the analysis of only a few cells isolated on a cell sorter or a few cells isolated via micro dissection from a glass slide after microscope identification and selection.
The solution to the problem is the use of a non-selective poly A mRNA labeling and amplification method, i.e. a method not encompassing cDNA synthesis.
The present invention provides such a method. The present invention is directed to a method for amplifying RNA by creating, in a non specific manner, multiple RNA copies starting from nucleic acid containing starting material comprising a pool of mRNA""s each mRNA comprising a poly-A tail, wherein the material is contacted simultaneously with an oligonucleotide comprising an oligo-dT sequence, the sequence of a promoter recognized by a RNA polymerase and a transcription initiation region which is located between the oligo-dT sequence and the sequence of the promoter, and further with an enzyme having reverse transcriptase activity, an enzyme having RNase H activity and an enzyme having RNA polymerase activity and the necessary nucleotides and the resulting reaction mixture is maintained under the appropriate conditions for a sufficient amount of time for the enzymatic processes to take place.
This will lead to the formation of multiple anti-sense RNA copies of the mRNA""s present in the reaction mixture. The method of the present invention does not involve the production of cDNA intermediates; RNA is copied directly from the mRNA present in the material under investigation. The method of the present invention does not need a cDNA as a basis for the amplification of the RNA. The RNA is synthesized by an RNA polymerase, directly from the mRNA template. The activity of the RNA polymerase is independent from any secondary structures present in the mRNA and thus there are no differences in the way the different mRNA""s are amplified depending on structures in the mRNA""s. The copies made represent the original mRNA population as present in the starting material.
The oligonucleotides used with the method of the invention comprise an oligo-dT sequence which will hybridize to the poly- and enylated tail at the 3xe2x80x2 end of the mRNA""s. The oligonucleotides further comprise the sequence of a promoter recognized by an RNA polymerase and a transcription initiation region which is located between the oligo-dT sequence and the sequence of the promoter. The promoter may be the promoter for any suitable RNA polymerase. Examples of RNA polymerases are polymerases from E. coli and bacteriophages T7, T3 and SP6. Preferably the RNA polymerase is a bacteriophage-derived RNA polymerase, in particular the T7 polymerase.
The oligonucleotide may be blocked at its 3xe2x80x2 end. If the oligonucleotide is not blocked at its 3xe2x80x2 end, it is extendible by the reverse transcriptase. However, the cDNA that would thus be generated will not be a part of the amplification mechanism (as it is with prior art methods). It even interferes with the other enzymatic reactions. The extension of the oligonucleotide is only with a very limited number of nucleotides because if the promoter is made double stranded the transcription on the mRNA template by the RNA polymerase will start immediately. This transcription will xe2x80x9cpushxe2x80x9d the RT from the RNA template and extension (i.e. cDNA synthesis) of the oligonucleotide can no longer occur. The oligonucleotide can be blocked at its 3xe2x80x2 end to prevent any extension therefrom by the reverse transcriptase along the RNA template; the reverse transcriptase will not be able to start extension of the 3xe2x80x2 end of the oligonucleotide and no cDNA is synthesized. The reverse transcriptase does synthesize a complementary strand of the promoter sequence present in the template. The use of the oligonucleotide is depicted schematically in FIG. 1. Upon hybridization of the oligonucleotide the poly-A sequence of the mRNA is cut by an enzyme having RNase H activity. This activity may be the RNase H activity of the reverse transcriptase or the RNaseH activity of a separate enzyme like, for example, E.coli RNaseH, or both. In that respect preferred transcriptases used with the method of the invention are transcriptases having RNaseH activity, such as AMV-RT or MMLV-RT. The newly generated 3xe2x80x2 end of the RNA is extended on the oligonucleotide template to generate a double stranded promoter sequence. By application of the RNA polymerase new RNA copies of the original mRNA are made. During this transcription step labels may be incorporated and typically 100-1000 copies of each RNA are being made. The copies made are antisense RNA and thus comprise a poly-T stretch at the 5xe2x80x2 end.
Since the RNA polymerase normally uses a double stranded template for the transcription the enzymes is not likely to be hampered by structures in the mRNA. Furthermore, the processivity of, for example, the T7 RNA polymerase is very high, usually more than 250 nucleotides per second on a DNA template. This means that the amplification rate is determined by the number of initiation events per promoter, per time unit. Since the promoter is identical for each mRNA there is no selectivity in the amplification.
The conditions under which the reaction, should be performed are the normal conditions, i.e. buffer concentrations and temperatures, known to be optimal for the mix of enzymes used.
If the interest exists to make an expression profile of just a few cells the above described amplification may not yield enough copies of the RNA, for example to generate a signal if the copies are labeled. In certain special cases the RNA may need to be amplified further without introducing selectivity, thus again avoiding i.e. cDNA synthesis. There are multiple solutions to this problem, all transcription based. An elegant solution is depicted in FIG. 2. The newly synthesized RNA may now be further amplified by the following method. To the 3xe2x80x2 end of every RNA molecule a double stranded promoter sequence is ligated by using RNA ligase. Since all 3xe2x80x2 ends are chemically identical there is no selectivity. The ligated promoter is used to initiate a second round of transcription generating more (labeled) RNA. This is illustrated in FIG. 2.
Thus in a preferred method of the invention the generated RNA copies made as described above are contacted with an RNA ligase, a double stranded nucleic acid complex comprising a double stranded DNA promoter sequence that can be recognized by a RNA polymerase, whereby one strand of said complex has a stretch of RNA attached to the 5xe2x80x2 end of one of the DNA strands, an enzyme having RNA polymerase activity, and the necessary nucleotides. The resulting reaction mixture is maintained under the appropriate conditions for a sufficient amount of time for the amplification to take place
Again, one or more of the nucleotides used may be labeled. Due to the orientation of the RNA polymerase promoter sequence the RNA template is used to generate new sense strand RNA molecules. Typically 100-1000 copies of each RNA is being made in the transcription reaction by the RNA polymerase.
Preferably the stretch of RNA attached to the 5xe2x80x2 end of one of the DNA strands is phosphorylated at the 5xe2x80x2 end. Phosporylation enables the 5xe2x80x2 end to be ligated.
The promoter may be the same as in the first part of the procedure for example, the T7-promoter sequence may be used and the RNA polymerase than is T7 RNA polymerase.
Interestingly the sense RNA made in this second round of transcription contains again a poly A stretch at the 3xe2x80x2 end making is possible to perform multiple cycles of amplification by repeatedly performing the method as illustrated by FIG. 1 and the method using the ligase as illustrated in FIG. 2.
The procedure wherein the ligase is used may be performed as a separate reaction. That is, after RNA copies have been generated in a procedure like the one depicted in FIG. 1, the RNA copies may be transferred to another reaction medium and subjected to the second reaction.
When all enzymes and the oligonucleotide and the promoter construct are combined with the initial reaction mixture a continuous process may even be obtained.
Another elegant method to further enhance the amplification factor of the non-biased mRNA amplification method is by adding a poly A nucleotide stretch to the 3xe2x80x2 end of the newly synthesized RNA. The poly nucleotide stretch is added by the enzyme poly A polymerase. To this added poly A sequence, the oligonucleotide, encompassing an oligo T stretch and T7 promoter, can hybridize again and the previously described process may take place again. As a result again RNA will be made by the transcription process and this newly synthesized RNA will be identical (for the large part) to the original mRNA that the whole reaction started with in the first place. One skilled in the art understands that the oligonucleotide, encompassing an oligo T stretch and T7 promoter can also hybridize again to this RNA and t he process may be considered a continuous process of RNA synthesis by transcription, oligonucleotide annealing and double strand promoter synthesis.
Thus in a preferred method of the invention the generated RNA copies made as described before in the basic method are contacted with a poly A polymerase, an oligonucleotide, encompassing an oligo T stretch and T7 promoter, a reverse transcriptase, a RNase H, a RNA polymerase and the necessary nucleotides. The resulting reaction mixture is maintained under appropriate conditions for a sufficient amount of time for the amplification to take place. In the mix one or more nucleotides used may be labeled.
Due to the position of the newly added poly A stretch (3xe2x80x2 end of the RNA molecules) the RNA polymerase will generate RNA of the opposite polarity. The oligonucleotide, encompassing an oligo T stretch and T7 promoter may be the same as in the first part of the procedure.
The procedure in which the poly A polymerase is added may be performed as a separate reaction. That is, after RNA copies have been made in a procedure like the one depicted in FIG. 1, the RNA copies may be transferred to another reaction medium and subjected to the reaction with poly A polymerase; starting a continuous amplification process.
When the poly A polymerase is added to the initial reaction mixture the continuous amplification process may even start immediately from the original mRNA template.